PERSISTENT COPURIFICATION OF ENZYMES CATALYZING THE SEQUENTIAL GLUCOSYLATION AND SULFATION STEPS IN GLUCOSINOLATE BIOSYNTHESIS

The last two steps in glucosinolate biosynthesis involve successively a glucosylation and a sulfation reaction, catalyzed by UDPglucose: thiohydroximate glucosyltransferase and PAPS: desulfoglucosinolate sulfotransferase. In an effort to obtain structural information on the individual enzymes, separation was attempted on numerous chromatographic and electrophoretic media. Successive ammonium sulfate fractionation, Mono Q ion-exchange chromatography and gel filtration of Brassica juncea L. cell extracts resulted in co-purification of the two enzymes. Subsequent resolution of the activities was unsuccessful by hydrophobic interaction chromatography, metal chelation chromatography and affinity chromatography on the following ligand bound gels: reactive dyes, uridine-5′-diphosphate, 3′-phosphoadenosine- 5′-phosphate, uridine-5′-diphosphate glucuronic acid and desulfobenzylglucosinolate. Both enzymes had similar electrophoretic mobility in native polyacrylamide gels and also co-eluted from a chromatofocussing column. The latter system pointed to a difference in stability as the loss of sulfotransferase activity was greater than of glucosyltransferase activity. Stability tests indicated that the glucosyltransferase was more stable below pH 7.5, while the sulfotransferase was more stable above pH 8.0. Furthermore, the sulfotransferase exhibited stability up to 40°C, while the glucosyltransferase was only stable up to 30°C. The above results suggest that the close similarities in chromatographic and electrophoretic properties may preclude separation of the two enzymes in their native form, but that thermodenaturation of the glucosyltransferase activity may advance the isolation of the sulfotransferase. © 1990, Gustav Fischer Verlag, Stuttgart. All rights reserved.